We found that propagation of different prions can be affected differently by the 97% identical and functionally interchangeable Hsp70 isoforms Ssa1 and Ssa2, and by our previously identified prion-impairing mutations in conserved Hsp70 residues. The ability to distinguish such differences between proteins that do not affect cell growth provides an acutely sensitive means to approach the problem of uncovering the underlying mechanisms. Using our system we discovered that nearly identical naturally occurring Hsp70 isoforms can influence propagation of different yeast prions in different ways. We are also working toward understanding the basis for these differences and for the exquisite functional specificity of natural Hsp70 isoforms.[unreadable] Our genetic work showed PSI+ prion propagation was weakened in cells lacking the Hsp70 Ssa1 but not in those lacking Ssa2. Conversely, we and others found URE3 prion propagation was normal in cells lacking Ssa1 but weakened in those lacking Ssa2. We also showed elevated expression of Ssa1 but not Ssa2 eliminated URE3 while neither excess Ssa1 nor Ssa2 affected PSI+. Additionally, we constructed a system for assessing function of any Hsp70 isoform in cell growth and prion propagation and used it to show that both can be supported by constitutively expressed, but not stress-inducible, mammalian and plant Hsp70s. Because Hsp70s have similar broad substrate specificity we hypothesize that functional differences are due to regulation of substrate binding rather than to specificity of substrate binding. By constructing and characterizing hybrid proteins we identified structural domains of interspecies, Ssa1 and Ssa2 isoforms that confer functional differences in growth and prion propagation. We are analyzing ATPase, substrate binding and protein folding capability of purified Ssa1 and Ssa2 to determine differences in enzymatic activities that confer the observed effects on prions in vivo. A variety of physical and fluorescence analyses of amyloid in strains with different prions that express the different Hsp70 isoforms and hybrids are being done to understand how the altered Hsp70 functions affect structural properties of amyloid in vivo.[unreadable] In related work we found that certain Hsp70 mutations have similar but non-identical effects on different prions. The basis of such differences could be mediated through a direct interaction of Hsp70 with the URE3 and PSI+ prion proteins (e.g. altered substrate binding affinity of Hsp70 for different prion proteins), or through overall effects on functions of chaperone machinery (e.g. different prions depend on different degrees or specificity of function of various chaperone machinery components). Using a genetic approach we identified and characterized many prion-impairing and second-site suppressing mutations in Ssa1. Genetic analyses of the prion-impairing Hsp70 mutants point to a common alteration in the Hsp70 reaction cycle as the basis for the anti-prion effects. We have purified several Hsp70s with prion-impairing mutations as well as those containing both forward and second-site suppressing mutations. Enzymatic analyses of the purified mutant proteins are being done to determine if the different mutations cause the same effect on Hsp70 activity or if altering different enzymatic activities could cause a similar alteration in Hsp70's reaction cycle leading to the same anti-prion effect.[unreadable] In addition to studying chaperones, our system has contributed to characterization of non-prion related functions of prion domains, and influence of prions in an unrelated translation system.